The endotoxins are lipopolysaccharides (LPS) of gram negative bacteria such as E.coli, and exist in the outer membrane of the cell envelope. They account for more than half the mass of the outer membrane of the cell envelope and they are constantly shed into the environment of the bacterium (Pearson 1985). The basic unit size of LPS is 10,000 to 20,000. However in aqueous solutions LPS generally exists in vesicles ranging in molecular weight from 300,000 to 1 million (Weary 1985).
The LPS molecule contains 3 distinct chemical regions, the Lipid A region, a central polysaccharide region and the O-antigen region. The Lipid A region resides in the cell membrane when endotoxins are contained within the cell wall. This is linked to a central polysaccharide core and this in turn is linked to the O-antigenic side chain, a repeating oligosaccharide structure which varies with different gram negative species.
The Lipid A region is composed of a glucosamine disaccharide containing phosphate groups and is highly substituted with long chain fatty acids. It is now known that Lipid A is responsible for most, if not all, activity associated with bacterial endotoxins and that endotoxins must be released from the bacterial surface to be effective (Rietschel and Brade 1992). The biological activities induced by endotoxins are extremely diverse. These are mediated through the activation of macrophages and other cellular components which lead to a wide range of biological effects. In mild doses, endotoxins produce moderate fever and stimulation of the immune system which in turn leads to microbial killing. In higher doses, they produce high fever, hypotension disseminated blood clotting and lethal shock.
The presence of endotoxins in biologically derived products (biologicals) prepared for therapeutic use is of major concern due to the diverse and potentially harmful biological activities of these molecules. Maintaining sterility in processes used in the manufacture of biologicals, together with stringent protocols for the preparation of equipment, helps to ensure products are free of endotoxins. However, raw materials used to manufacture biologicals are often not sterile. Indeed, when the source of a biological is from a gram negative bacterial culture (e.g. a method using an E.coli fermentation system to express recombinant protein), the endotoxin levels in the starting material will be very high. In practice, maintenance of sterility throughout an entire process is not always possible or cost effective. Therefore it is often desirable to have methods in place which either destroy or remove endotoxins while maintaining the integrity of the therapeutic biological component.
There have been numerous approaches to achieving destruction or removal of endotoxins (Pearson 1985, Weary 1985). These include hydrolysis with acid or base, oxidation, alkylation, heat treatment and treatment with polymicin B. However with each of these approaches the effect of the inactivation method on the desired biological product must be evaluated. Furthermore, while pyrogenic activity may be reduced, often endotoxin components remain and the presence of these endotoxin components may be of no benefit in the final product and could possibly be detrimental. It is therefore preferred to remove these endotoxin components from the final biological product.
Selective binding of endotoxins on charged, hydrophobic or affinity media, or separation on the basis of size can be performed. At pH levels greater than pH2, endotoxin aggregates are negatively charged and will bind to positively charged surfaces such as asbestos or anion exchangers (Weary 1985). Endotoxins will also bind to aliphatic polymers such as polypropylene, polyethylene, polyvinylidene fluoride, polytetrafluoroethylene and hydrophobic chromatographic systems via hydrophobic interactions. Endotoxins can also be specifically removed by affinity chromatography using immobilised polymicin B. Additionally, because endotoxins exist primarily as large molecular weight complexes, they can often be removed from desired components by ultrafiltration or gel filtration methods.
Each of the above mentioned procedures presents a problem. Biological molecules, such as human therapeutic proteins derived from plasma, will in general be positively charged at low pH (i.e. less than pH 4). Although endotoxins are negatively charged at relatively low pH and thus will bind to positively charged resins, many therapeutic proteins are unstable under these conditions. Furthermore, complete resolution between protein and endotoxins cannot always be effected. Hydrophobic chromatographic systems will effectively bind endotoxins but often will also bind the desired biological molecule. Additionally, these hydrophobic chromatographic systems can be difficult to regenerate. Affinity chromatography systems using polymicin B are expensive in terms of media cost. Furthermore, in such systems the support can be difficult to regenerate resulting in a short life for this matrix. Size exclusion chromatography or ultrafiltration can also be used to reduce endotoxin levels. However size exclusion chromatographic systems and ultrafiltration systems will only be useful when there is a substantial size difference between the target biological molecule and the endotoxin molecule. Additionally, size exclusion chromatographic systems suffer from the problem of limited capacity.
The major difficulty in separating endotoxins from proteins lies in designing a support material that exhibits a high specificity for endotoxins but a low specificity for proteins. The ideal support
should not interact with proteins PA1 should exhibit a high capacity for endotoxins PA1 should be able to be regenerated PA1 should be stable under conditions of operation, including regeneration methods PA1 should be acceptable for use in the manufacture of therapeutic products. PA1 (a) plasma (e.g. albumin, immunoglobulins, clotting factors, protease inhibitors and growth factors); PA1 (b) recombinant or cell culture expression systems (e.g. human growth hormone, interferons, cytokines, insulin monoclonal antibodies); PA1 (c) fermentation systems used in the manufacture of vaccines (e.g. components from bordetella pertussis, cultures used in whooping cough vaccine).
In work leading to the present invention, it has been found that particular chromatographic gel matrices which have in the past been manufactured and used for gel filtration meet the above criteria, exhibiting a minimal interaction with proteins and a high affinity for endotoxins. Furthermore, it has been established that these matrices are stable under the operational and regeneration systems that have developed for binding of endotoxins and for eluting the bound endotoxin.